The vascularization of tissue constructs remains a major challenge in regenerative medicine. Without its own blood supply, an engineered construct relies mainly on diffusional oxygen supply, which can only support a thin layer of viable tissue. Therefore, vascularization of a tissue construct is crucial for its successful implantation, survival, and integration with the host tissue. The formation of mature and functional vascular networks requires interaction between endothelial cells (ECs) and vascular smooth muscle cells (v-SMCs). During early vascular development, ECs line the vessel wall and organize into an immature vasculature. To further stabilize these nascent vessels, ECs secrete platelet-derived-growth-factors (PDGF) to induce the differentiation of specialized mesenchymal stem cells (MSCs) into pericytes in capillaries or SMCs in larger vessels. At this later stage, transforming growth factor-beta 1 (TGF-β1) regulates vessel maturation by inducing v-SMC differentiation and the generation of extracellular matrix (ECM) molecules, such as collagen, fibronectin, and Laminin. Embedded within this ECM, v-SMCs provide physical support to the vasculature and aid in the maintenance of endothelial viability. This process of vascular morphogenesis involving ECs interacting with both the ECM and v-SMCs has been widely studied in vitro using Matrigel assays. When grown on Matrigel, a basement membrane matrix enriched with laminin, ECs and v-SMCs interact to form capillary-like structures (CLSs) that resemble tube formation in vivo. Thus, v-SMCs are key components in engineering vascularized tissue.
One major limitation of this therapeutic approach has been the lack of a reliable source of v-SMCs. Since v-SMCs isolated from patients are usually derived from diseased organs that have limited proliferative capacity and reduced collagen production, they have impaired mechanical strength and cannot support vascular function. Alternatively, bone marrow-derived MSCs have been used to engineer small-diameter vessel grafts and blood vessels which are stable and functional in vivo. Adipose tissue and neural crest tissue also contain populations of multipotent cells that can be differentiated into functional v-SMCs. Another promising source of v-SMCs is human embryonic stem cells (hESCs), which are pluripotent, have high proliferative capacity, exhibit low immunogenicity, and have been shown to repair ischemic tissues and restore blood flow (Sone et al. (2007) Arterioscler Thromb Vasc Biol 27, 2127-34). Studies demonstrating the derivation of v-SMCs from embryonic or pluripotent induced stem cells (human or mouse) have utilized various approaches to guide differentiation—such as coculture on OP9 feeder layer or retinoic acid supplementation—and to purify derivatives by sorting for specific vascular progenitors or mature markers, selecting for stable expression of SMC promoter, or isolating the outgrowth of embryoid bodies (EBs). In previous studies, we have demonstrated that the derivation of vascular lineages from hESCs can be achieved by administration of angiogenic growth factors, either by monolayer, two-dimensional (2D) differentiation protocol, or by isolation of vascular progenitor cells or CD34+ cells from 10-day old EBs, followed by selective induction into either endothelial like cells (using vascular endothelial growth factor; VEGF) or smooth-muscle-like cells (SMLCs; using PDGF-BB).
There is a need to develop simple procedure that results in highly purified cultures of SMLCs which are mature enough to exhibit characteristics such as contractile phenotypes and the ability to support vasculature in vitro.